The field of this invention is a herpes virus entry receptor (HVEM). More particularly, the field of the present invention is recombinant mammalian HVEM, polynucleotides encoding that HVEM, and methods of making recombinant HVEM.
Glycosaminoglycan chains on cell surface proteoglycans serve as receptors for the binding of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) to cells. Binding is not sufficient for entry, however: other cell surface components are necessary for virus entry, which occurs by fusion of the virion envelope with a cell membrane. For example, Chinese hamster ovary (CHO) cells express glycosaminoglycan chains to which HSV-1 and HSV-2 can bind, but are resistant to the entry of some HSV strains, particularly HSV-1(KOS).
The present invention is directed to a newly discovered protein that enables herpes simplex virus (HSV) to penetrate into cells and is a previously undiscovered member of the family of receptors designated the tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR) family. Members of this family have characteristic repeats of amino acid sequence containing multiple cysteines and serve as receptors for a variety of specific ligands, including but not limited to cytokines. The protein is designated herpes virus entry receptor protein or HVEM.
By identifidng the gene that encodes HVEM, by showing that HVEM can mediate the entry of HSV into cells and by performing experiments to define viral and cell factors that influence the ability of HVEM to mediate HSV entry, the inventors have provided the knowledge and biological material required (i) to develop antiviral drugs that can act to block HSV (and perhaps other herpesvirus) entry into cells; (ii) to identify other members of the TNFR/NGFR family (or other cell surface molecules) that can serve as receptors for HSV-1, HSV-2 or other herpesviruses; (iii) to identify the natural ligand for the receptor, and (iv) to develop therapeutic approaches for enhancing or inhibiting action of the ligand on the receptor, depending on the pathologic or, beneficial consequences of this action.
In one aspect, the present invention provides an isolated and purified polynucleotide comprising a nucleotide sequence consisting essentially of the nucleotide of SEQ ID NO:1 from about nucleotide position 294 to about nucleotide position 1142; (b) sequences that are complementary to the sequences of (a), and (c) sequences that, when expressed, encode a polypeptide encoded by a sequence of (a). A preferred polynucleotide is a DNA molecule. In another embodiment, the polynucleotide is an RNA molecule. A preferred polynucleotide is SEQ ID NO:1.
In another embodiment, a DNA molecule of the present invention is contained in an expression vector. The expression vector preferably further comprises an enhancer-promoter operatively linked to the polynucleotide. In an especially preferred embodiment, the DNA molecule has the nucleotide sequence of SEQ ID NO:1 from about nucleotide position 294 to about nucleotide position 1142.
In another aspect, the present invention provides an oligonucleotide of from about 15 to about 50 nucleotides containing a nucleotide sequence of at least 15 nucleotides that is identical or complementary to a contiguous sequence of a polynucleotide of this invention. A preferred oligonucleotide is an antisense oligonucleotide that is complementary to a portion of the polynucleotide of SEQ ID NO:1.
The present invention also provides a pharmaceutical composition comprising a polypeptide or an antisense oligonucleotide of this invention and a physiologically acceptable diluent.
In another aspect, the present invention provides an HVEM polypeptide of mammalian origin. In one embodiment, that HVEM is an isolated and purified polypeptide of about 300 amino acid residues and comprises the amlo acid residue sequence of SEQ ID NO:2. More preferably, an HVEM of the present invention is a recombinant human HVEM.
In another aspect, the present invention provides a process of making HVEM comprising transforming a host cell with an expression vector that comprises a polynucleotide of the present invention, maintaining the transformed cell for a period of time sufficient for expression of the HVEM and recovering the HVEM. Preferably, the host cell is an eukaryotic host cell such as a mammalian cell or a bacterial cell An especially preferred host cell is a mammalian ovarian cell. The present invention also provides an HVEM made by a process of this invention. A preferred such HVEM is recombinant human HVEM.
The present invention still further provides for a host cell transformed with a polynucleotide or expression vector of this invention. Preferably, the host cell is a mammalian cell such as an ovarian cell.